1. Field of the Invention
The present invention relates to methods of augmenting protein synthesis. More specifically, the present invention relates to healing wounds by augmenting protein synthesis.
2. Description of Related Art
Wound healing is a complex process involving inflammation, recruitment of fibroblasts and macrophages, synthesis of collagen, and remodeling of the newly formed wound substrate.(4) Wound healing is believed to proceed at near maximal levels in the uncompromised host; however, numerous disease states and therapeutic interventions are associated with significant alterations in wound healing. Steroid, chemotherapeutic agents, diabetes, and ischemia all result in a reduction in collagen synthesis and decreased wound strength. Recent investigations have suggested that optimization of growth factor delivery in these problematic wounds may improve clinical outcome.
Naturally produced substances have been discovered which promote repair, healing and augmentation of tissues and organs. Such substances have been termed “growth factors”. Growth factors, usually proteins, initiate programs of differentiation and/or development within an organism.
When referring to tissue repair, the appellation “growth factor” is a misnomer. Confusion in separating the biological processes of growth from the processes involved in repair, healing and augmentation is often caused by the use of the term “growth factors” to describe these proteins. Repair, healing and augmentation, as discussed in detail below, are distinct biological activities and are clearly distinguishable from growth. Growth in the biological sense is defined as progressive development from a lower or simpler to a higher or more complex form of organization. Tissues and organs “grow” from a few similar appearing cells to a complex organized structure, such as a kidney or an eye. For clarity, organs are defined as functional units of the body containing multiple cell types. Examples of organs include, for instance, kidneys, eyes, the liver, the heart, bone, skin and cartilage. Tissues are defined as functional units of the body that are made up of almost an entirely single cell type. For instance, connective and support tissues are derived from and comprised of almost entirely a single cell type, e.g. fibroblast or muscle cell.
Growth factors can stimulate wound healing. The process of wound healing begins immediately following surface lesion or after skin proteins become exposed to radiation, chemical damage or extreme temperatures. Wound healing requires close control of degradative and regenerative processes, involving numerous cell types and complex interactions between multiple biochemical cascades. Growth factors released in the traumatized area stimulate and promote the following: 1) cell migration into the wound area (chemotaxis); 2) proliferation of epithelial cells, muscle cells, endothelial cells, blood cells and fibroblasts (mito-genesis); 3) formation of new blood vessels (angiogenesis); and 4) matrix formation and remodeling of the affected region including re-epithelization by keratinocytes. Studies on animals have shown that exogenously added growth factors can accelerate the normal healing process, and studies on humans have shown that growth factors can heal previously incurable wounds. Factors capable of enhancing wound healing are particularly important in treatment of patients with chronic wounds which may require daily treatment, represent a constant source of pain to the patient, may lead to life threatening infection and are a significant medical expense. Chronic wounds are those which are slow-healing or which do not heal at all and are common to diabetics, cancer patients and those confined to bed for long periods of time. Treatment of chronic wounds may consume up to $4 billion per year in medical expenses in the United States alone.
Despite their beneficial effect on bone, cartilage, skin and connective and support tissue, the use of growth factors poses several problems. Growth factors, when systemically administered, affect non-target organs and may therefore elicit a variety of adverse side effects. For instance, one recent article expressed the opinion that TGF-β may contribute to the renal lesions found in glomerulone-phritis, the leading cause of kidney failure in people with diseases such as lupus, diabetes and hypertension. Skerrett (1991). Further problems with growth factors are their instability and tendency to break down once purified and stored for therapeutic use. Moreover, many of the amino acid sequences of growth factors vary between species and are consequently recognized as foreign by dissimilar, or heterologous, species. There is the constant danger of eliciting an immune response upon administration of heterologous growth factors. Furthermore, there is no evidence that parenterally administered growth factors target bone, cartilage, skin, and connective and support tissues. Parenteral administration refers to intravenous, intramuscular, intraperitoneal and subcutaneous administration.
As proteins, growth factors are not suitable for oral administration, since they are digested and destroyed before entering the blood stream. Growth factors cannot be satisfactorily administered as topical ointments except for skin wounds, because they are only slowly absorbed by the body and subsequently break down rapidly. Because of these and other problems, growth factors are typically administered intravenously. Since naturally occurring growth factors can after the function of many organs and tissues of the body, intravenous administration of growth factors affects many non-target organs. A therapeutically effective compound that directly targets bone, cartilage, skin and connective and support tissues when parenterally administered or that can be directly applied to the tissues or organs that need to be repaired, healed or augmented is highly preferred to currently available naturally occurring growth factors.
Wound healing is in large part mediated by growth factors that control cellular migration into the wound area or synthesis of wound structural or regulatory proteins.(3) EGF is a 53 amino acid polypeptide that acts as a chemotactic factor for keratinocytes, vascular smooth muscle cells and granulation derived fibroblast.(2) Application of EGF results in accelerated wound healing as determined by an increase in tensile strength of the wound in normal animals.(6) 
Gene therapy using particle bombardment of nucleic acid-laden microcarriers with a gene gun allows intracellular delivery of DNA or RNA.(13) Such biolistic delivery is well suited for applications in wound healing where the application site is accessible.(6) DNA gene therapy has significant risks including insertional mutagenesis and uncontrolled promoter activity and promoter reactivation. Additionally, the problems detailed above have prevented others from attempting more detailed studies into the use of mRNA in this form of gene therapy.
Biolistic delivery and expression of a human EGF gene construct resulted in accelerated wound healing.(1) In this study an external sealed fluid filled wound chamber was used to protect the wound. Wounds treated with the human EGF plasmid pWRG1630 exhibited a 190-fold increase of EGF in the wound fluid and healed 20% faster than controls. EGF levels remained elevated for more than 8 days. It is noteworthy that the human EGF in vitro transcription vector created for these studies was derived from plasmid pWRG1630 therefore the EGF protein produced should have similar biological activity.
For wound healing applications, RNA mediated gene transfer is desirable as it avoids promoter expression uncertainty, and provides for a potent biologic effect for a finite therapeutic period without concerns of long-term deleterious effects. With the RNA delivery approach, target cells serve as a bioreactor for protein synthesis eliminating protein processing and modification difficulties noted with exogenously produced, recombinant products.(5) The mRNA delivery technique allows the use of more potent cellular factors or stimulants than previous possible as it is not associated with long term mutagenic concerns.(11) 
Translation of mRNA is now recognized as a key regulatory step in gene expression. Initiation of translation is the rate-limiting step and therefore a major regulatory target. The eukaryotic initiation factor family eIF, binds to the ribosome subunit facilitating protein translation. The rate of protein synthesis of eukaryotic cellular mRNA is controlled by the initiation step of translation because the translation initiation factor eIF4E is rate limiting.(8) The activity of eIF4E is regulated by phosphorylation that is acted on by various growth factors.
Over-expression of eIF4E is associated with aberrant growth and morphology in HeLa cells and malignant transformation of NIH T3T cells.(7, 10) Moreover, increased levels of eIF4E have been noted in carcinoma specimens. This raises the possibility that chronic over-expression of eIF4E may be oncogenic therefore potential therapeutic applications of eIF4E gene therapy have not been suggested.
Not all mRNA transcripts are translated with equal efficiency due to structural constraints in the 5′ untranslated region. These “weak” transcripts are hypothesized to be more dependent on eIF4E for translation, and their translational yield more responsive to increases in active eIF4E. Highly structured mRNAs that may be subject to this regulation include ones that encode growth factors (PDGF-b, ILGF-II, FGF-2, TGF-β, and VEGF), transcription factors (Ick, c-mos) and cell cycle regulators (CDK, p53).(9) A few growth-promoting proteins have been demonstrated to be regulated by the level of active eIF4E (cyclin D1, omithine decarboxylase and P23). For this invention, it is important to note that EGF is not predicted to have significant secondary structure in its 5′ untranslated region, and regulation of EGF by eIF4E has never been suggested, especially in light of the potential oncogenic effects.
Referring specifically to TGF-β, this growth factor belongs to a family of growth factors that produce multiple biological effects, including mitogenesis, growth regulation, regulation of cartilage and bone formation, chemotaxis and induction or inhibition of cell differentiation, depending on the tissue or cell type and the presence or absence of other growth factors. Most of the published work on TGF-β relates to its wound healing capabilities. However, TGF-β plays other physiological roles, as shown by the fact that it is known to be contained and produced within bone. Seyedin et al., “Cartilage-Inducing Factor”, J. Biol. Chem., 261: 5693–5695 (1986); and Robey et al., “Factor-Type .beta. (TGF-β) in vitro”, J. Cell Biol., 105: 457–463 (1987). TGF-β will enhance bone formation. Sporn et al., “Some Recent Advances in the Chemistry and Biology of Transforming Growth Factor-.beta.”, J. Cell Biol., 105: 1039–1045 (1987). Other members of the TGF-β family of growth factors, notably BMP, have also been shown to enhance bone formation. Wozney et al., “Novel Regulators of Bone Formation: Molecular Clones and Activities”, Science, 242: 1528–1533 (1988).
With specific regard to bone resorption, recent studies with purified cell membranes have shown that gallium nitrate (Ga(NO3)3) can block the transport of hydrogen atoms across osteoclast cell membranes. This hydrogen atom transport would otherwise lead to the dissolution of the mineral matrix of bone, thereby releasing calcium ions into the blood. Although TGF-β affects bone repair, healing and augmentation, it has not been shown to block transport of hydrogen atoms across osteoclast cell membranes. In fact, TGF-β has not been demonstrated to be a clinically effective antiresorptive agent capable of preventing accelerated bone breakdown and disordered calcium homeostasis. Indeed, unlike previously shown activity of gallium nitrate, TGF-β inhibits the differentiation and proliferation of osteoclastic cells, leading to decreased osteoclast cell numbers. Chenu et al., “Transforming Growth Factor Beta Inhibits Formation of Osteoclast-Like Cells in Long-Term Human Marrow Cultures”, Proc. Natl. Acad. Sci. USA, 85: 5683–5687 (1988). By contrast, rats treated with gallium nitrate have normal or increased numbers of osteoclasts. Cournot-Witmer et al., “Bone Modeling in Gallium Nitrate Treated Rats”, Calcif. Tis. Int., 40: 270–275 (1987).
There is no direct relationship between the deposition of the mineral component of bone and biologic bone repair, healing and augmentation. The mineral component of bone is made up of hydroxyapatite, a crystalline, inorganic complex of calcium and phosphate. Hydroxyapatite crystals “grow” in size in the physical process of accretion (i.e., addition) of new atoms of calcium and phosphate. Calcium accretion onto crystalline hydroxyapatite of bone is a passive physical-chemical process that does not require living cells. The synthesis of new matrix components, which requires living cells, the activation of specific genes and the de novo synthesis of proteins from organic elements, is unrelated to calcium accretion. The basic building blocks for matrix synthesis come from living cells and have, for the most part, been synthesized de novo by those cells. Disorders of calcium homeostasis, therefore, affect only the inorganic matrix of bone and are unrelated to repair, healing and augmentation in the biologic sense. Mechanisms involved in repair, healing and augmentation of the organic matrix of bone, cartilage, skin and connective and support tissues represent biologic processes that are different and distinct from mechanisms involved in calcium accretion.
Several pharmaceutical agents, including cisplatin, mithramycin, calcitonin, and bisphosphonates, have been shown to inhibit resorption of bone mineral matrix. None of these agents, however, have a proven beneficial effect on bone formation or wound healing. Cisplatin and mithramycin are cytotoxic agents which, when injected parenterally, act by killing the cells responsible for tissue breakdown, as well as those responsible for tissue formation. Calcitonin, a naturally produced hormone, transiently inhibits the activity of bone-resorbing cells (osteoclasts) to prevent bone breakdown. Calcitonin increases excretion of calcium by the kidneys and thus accelerates calcium loss from the body.
Bisphosphonates are a class of synthetic compounds that inhibit bone resorption. Etidronate (EHDP) is currently the only bisphosphonate approved for use in the United States. Osteoporosis patients who have been treated with EHDP, however, have shown a 50% increase in vertebral fracture rates in the third year. See, e.g., “Update: Bisphosphonates Editronate evaluated by FDA”, Lunar News, March 1991. The possible ineffectiveness of EHDP over long-term treatment tends to indicate that agents that inhibit bone resorption do not strengthen bone in a clinically significant manner, and in fact, may tend to weaken bone. Further, EHDP inhibits matrix-forming cells. Schenk et al., “Effect of Ethane 1-hydroxy-1,1-diphosphate (EHDP) and Dichloromethylene Diphosphonate (Cl2 MDP) on the Calcification and Resorption of Cartilage and Bone in the Tibial Epiphysis and Metaphysis of Rats”, Calcif. Tis. Res., 11: 196–214 (1973).
Fluoride-containing salts have been extensively tested for their effects on matrix-forming cells. Treatment with fluoride, however, results in the production of a highly abnormal (woven-type) bone matrix structure. Such fluoride-induced bone is weaker than normal bone. Jowsey et al., “Some Results of the Effect of Fluoride on Bone Tissue in Osteoporosis”, J. Clin. Endocrinol., 28: 869–874 (1968). Indeed, a recently completed study showed that fluoride did not significantly reduce skeletal fractures in osteoporotic women. Kleerekoper et al., “Continuous Sodium Fluoride Therapy Does Not Reduce Vertebral Fracture Rate in Postmenopausal Osteoporosis”, J. Bone and Min. Res., 4:S376 (1989).
Estrogen replacement therapy has resulted in increased bone mass in estrogen-deficient, post-menopausal women. Lindsay et al., “Long-Term Prevention of Postmenopausal Osteoporosis by Estrogen Treatment”, Lancet, 1: 1038–1041 (1976). Estrogen directly affects bone-forming cells to increase matrix elements, such as collagen, and to increase an endogenous growth factor, insulin-like growth factor-I (IGF-1). Ernst et al., “Estradiol Effects on Proliferation, Messenger RNA for Collagen and Insulin-like Growth Factor-I, and Parathyroid Hormone-Stimulated Adenylate Cyclase Activity on Osteoblastic Cells from Calvariae and Long Bones”, Endocrinol., 125: 825–833 (1989). However, the benefits of estrogen treatment are limited to perimenopausal women, those women who are about to enter or who have entered menopause. Furthermore, estrogen treatment is associated with increased risk of uterine and breast cancer. Bergkvist et al., “The Risk of Breast Cancer After Estrogen and Estrogen-Progestin Replacement”, N. E. J. Med., 321: 293–297 (1989).
In summary, exogenous growth factors, while capable of inducing synthesis of new matrix components in a manner that simulates natural, normal, conditions of repair, healing and augmentation of organs and tissues, have proven to be difficult to administer and tend to cause side effects. Further, various pharmaceutical agents have proven unsuccessful in inducing synthesis of new matrix components in a manner that simulates natural, normal, conditions of repair, healing and augmentation of organs and tissues.
It would therefore be useful to develop biolistic delivery mechanisms for delivery of mRNA to a wound, or other site in need of transient increased protein synthesis, for increased cellular translation of endogenous mRNA to augment wound healing.